Numerous commercially valuable plants, including common agricultural crops, are susceptible to attack by insect and nematode pests, causing substantial reductions in crop yield and quality. For example, growers of maize (Zea mays), commonly referred to as corn in the United States, face a major problem with combating pest infestations. Insects, nematodes, and related arthropods annually destroy an estimated 15% of agricultural crops in the United States and an even greater percentage in developing countries. In addition, competition with weeds and parasitic and saprophytic plants account for even more potential yield losses. Yearly, such pests cause over $100 billion in crop damage in the United States alone.
In an effort to combat pest infestations, various methods have been employed in order to reduce or eliminate pests in a particular plot. These efforts include rotating corn with other crops that are not a host for a particular pest and applying pesticides to the above-ground portion of the crop, applying pesticides to the soil in and around the root systems of the affected crop. Traditionally, farmers have relied heavily on chemical pesticides to combat pest damage. However, the use of chemical pesticides is costly, as farmers apply billions of gallons of synthetic pesticides to combat these pests each growing season, costing nearly $8 billion. In addition, such pesticides are inconvenient for farmers, result in the emergence of insecticide-resistant pests, and they raise significant environmental and health concerns.
Because of concern about the impact of pesticides on public health and the health of the environment, significant efforts have been made to find ways to reduce the amount of chemical pesticides that are used. Recently, much of this effort has focused on the development of transgenic crops that are engineered to express insect toxicants derived from microorganisms. For example, U.S. Pat. No. 5,877,012 to Estruch et al. discloses the cloning and expression of proteins from such organisms as Bacillus, Pseudomonas, Clavibacter and Rhizobium into plants to obtain transgenic plants with resistance to such pests as black cutworms, armyworms, several borers and other insect pests. Publication WO/EP97/07089 by Privalle et al. teaches the transformation of monocotyledons, such as corn, with a recombinant DNA sequence encoding peroxidase for the protection of the plant from feeding by corn borers, earworms and cutworms. Jansens et al., Crop Sci., 37(5):1616-1624 (1997), reported the production of transgenic corn containing a gene encoding a crystalline protein from Bt that controlled both generations of Eastern Corn Borer (ECB). U.S. Pat. Nos. 5,625,136 and 5,859,336 to Koziel et al. reported that the transformation of corn with a gene from Bt that encoded for a δ-endotoxin provided the transgenic corn with improved resistance to ECB. Additionally, a comprehensive report of field trials of transgenic corn that expresses an insecticidal protein from Bt has been provided by Armstrong et al., Crop Science, 35(2):550-557 (1995). For these and other reasons, there is a demand for alternative insecticidal agents for agricultural crops. For example, maize plants incorporating transgenic genes which cause the maize plant to produce insecticidal proteins providing protection from the target pest(s) is a more environmentally friendly approach to controlling pests. The use of pesticidal crystal proteins derived from the soil bacterium Bt commonly referred to as “Cry proteins” have been utilized. Cry proteins are globular protein molecules which accumulate as protoxins in crystalline form during late stage of the sporulation of Bt. After ingestion by the pest, the crystals are solubilized to release protoxins in the alkaline midgut environment of the larvae. Protoxins (˜130 kDa) are converted into mature toxic fragments (˜66 kDa N terminal region) by gut proteases. Many of these proteins are quite toxic to specific target insects, but harmless to plants and other non-targeted organisms. Some Cry proteins have been recombinantly expressed in crop plants to provide pest-resistant transgenic plants. Among those, Bt-transgenic cotton and corn have been widely cultivated.
A large number of Cry proteins have been isolated, characterized and classified based on amino acid sequence homology. See Crickmore et al., Microbiol. Mol. Biol. Rev., 62:807-813 (1998). This classification scheme provides a systematic mechanism for naming and categorizing newly discovered Cry proteins. Bt toxins have traditionally been categorized by their specific toxicity towards specific insect categories. For example, the Cry1 group of toxins is toxic to Lepidoptera, and includes, but is not limited to, Cry1Aa, Cry1Ab and Cry1Ac. See Hofte et al., Microbiol. Rev., 53:242-255 (1989). The Cry1 classification is the best known and contains the highest number of cry genes, currently totals over 130. Cry1 and Cry2 proteins share a minimal amount of sequence homology. See, e.g., Crickmore et al. (1998) indicating that Cry1A and Cry2A classes are among the most divergent.
It has generally been found that individual Cry proteins possess relatively narrow activity spectra. For example, Cry1Ac was the first toxin to be deployed in transgenic cotton for control of H. virescens and H. zea insect pests. This toxin is known for its high level toxicity to H. virescens. However, it is slightly deficient in its ability to control H. zea and has almost no activity on Spodoptera species. Additionally, Cry1Ab toxin has slightly less activity on H. zea than Cry1Ac but has far superior activity against S. exigua. 
Cry2A is an exception as it is unusual in that this subset of Cry proteins possesses a broader effective range that includes toxicity to both the Lepidoptera and Diptera orders of insects. The Cry2A protein was discovered to be a toxin showing a dual activity against Trichoplusia ni (cabbage looper) and Aedes taeniorhynchus (mosquito) (Yamamoto & McLaughlin, Biochem. Biophys. Res. Comm., 130:414-421 (1982)). The nucleic acid molecule encoding the Cry2A protein (termed Cry2Aa) was cloned and expressed in B. megaterium and found to be active against both Lepidoptera and Diptera insects (Donovan et al., J. Bacteriol., 170:4732-4738 (1988)). An additional coding sequence homologous to Cry2Aa was cloned (termed Cry2Ab) and was found to be active only against Lepidoptera larvae (Widner & Whiteley, J. Bacteriol., 171(2):965-974 (1989)).
Second generation transgenic crops could be more resistant to insects if they are able to express multiple, novel and/or synergistic Bt genes.
Accordingly, it is an objective of embodiments of the present invention to provide synergistic resistance to plant insects.
Another objective of embodiments of the invention includes methods for incorporating multiple Cry proteins into transgenic plants, namely maize.